CN104198613A - Method for analyzing protein O-glycosylation sites - Google Patents
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Abstract
The invention relates to a method for analyzing protein O-glycosylation sites. The method comprises the following steps: (1), sequentially carrying out endonuclease digestion, endoglycosidase and exoglycosidase digestion on a proteome sample or a single protein sample to prepare a peptide and non-glycopeptide protein sample or single protein sample with a GalNAc label; (2) loading the sample on a durian agglutinin chromatographic column and separating to prepare a proteomic sample; (3) separating the single protein sample or the proteomic sample through with a C18 reversed-phase chromatographic column, and then detecting by a high-definition mass spectrometer in a cation mode to obtain a high-definition mass spectrogram; (4) processing the obtained mass spectrometric data and obtaining protein glycosylation site information though the searched result. The site information can be more rapidly, more accurately and more comprehensively obtained through establishing the method for detecting the O-glycosylation sites of glycoproteins in the single protein sample and the proteomic sample.
Description
Technical field
The present invention relates to a kind of method of analyzing proteins O-glycosylation site, be particularly related to and a kind ofly adopt restriction endonuclease and excision enzyme Partial digestion sugar chain and combine the method that LC-MS technology detects and identifies glycoprotein O-glycosylation site, belong to technical field of biotechnology.
Background technology
The glycosylation of albumen is a kind of important posttranslational modification.Mainly contain N-glycosylation (N-glycosylation) and two kinds of forms of O-glycosylation (O-glycosylation).Protein glycosylation has participated in a lot of processes of cell, and plays the part of and have important role.In addition, the generation evolution of the some diseases such as cancer is often accompanied by the ANOMALOUS VARIATIONS of glycosylation site and the sugar chain of albumen.Therefore the ANOMALOUS VARIATIONS that, the analysis of glycosylation site discloses glycoprotein for research is most important.Locus Analysis in Shoots not only can obtain the glycosylated change information of mark of special disease intuitively, also can make the further sugar chain structure locus specificity analysis direct convenience more that becomes.
Because N-glycosylation occurs in protein characteristic sequence, there is common pentasaccharides core texture, and there is the general endo-glycosidase that discharges N-sugar chain from protein, therefore, the analytical approach of albumen N-glycosylation site is very ripe.By contrast, the analysis of O-glycosylation site has great challenge.O-glycosylation occurs on serine or threonine, does not have feature modification sequence can do decorating site prediction; The structure of O-sugar chain is more complicated, at least has 8 kinds of core textures; And, the general restriction endonuclease that also discovery can discharge O sugar chain from albumen, these reasons make the analysis of O-glycosylation site difficult.
Conventionally the analysis meeting of O-glycosylation site is used chemical method for releasing as β-null method (Zheng Y, et al.Talanta2009,78:358-363), but chemical method is difficult to control sometimes, can produce some subsidiary reactions.Outside upper method, also have other some excision enzyme methods (
p et al.J Proteome Res, 2007,6:3021-3031), identify that the O-glycosylation site obtaining is more limited.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, the circumscribed enzyme process analyzing proteins of a kind of mixing O-glycosylation site is provided, can be used for the analysis of the disease biomarker glycosylation sites such as glycoprotein.
Summary of the invention
The present invention utilizes restriction endonuclease to cut N sugar chain and O-sugar chain with mixing excision enzyme, and the asparagine that connects N-sugar chain becomes aspartic acid, and the serine or the threonine that connect O-sugar chain only leave a GalNAc sugar label.The peptide section of single albumen adopts LC/MS/MS Direct Analysis, and the glycosylated peptide segment molecule of O-amount occurs increases 203Da, by high resolution mass spectrum, obtains accurate molecular weight and secondary fragment information, finds out accordingly O-glycosylation site.Imitate this O-glycosylation analysis of group, first use durian agglutinin by the glycopeptide enrichment of Qie Shengyige GalNAc unit, thereby loci is analyzed.The method is simple, and fast, high flux, can be for the detection of the glycosylation site of standard model and actual sample.
Detailed Description Of The Invention
A method for analyzing proteins O-glycosylation site, step is as follows:
(1) by protein group sample or single protein sample after endonuclease digestion, again through endoglycosidase and exoglycosidase combination excision N sugar chain and part O-sugar chain, through drying under reduced pressure, make with the GalNAc sugar peptide section of label and the group protein sample of non-glycopeptide or single protein sample;
(2) what step (1) is made is splined on durian agglutinin chromatographic column with the GalNAc sugar peptide section of label and the group protein sample of non-glycopeptide and carries out separated, after collecting enzyme and cutting with the peptide section solution of GalNAc sugar label, after drying under reduced pressure, make group and learn protein sample;
(3) the group protein sample that single protein sample step (1) being made or step (2) make is used C
18reverse-phase chromatographic column is separated, then under positive ion mode, with high-resolution mass spectrometer, detects, and obtains high resolution mass spectrum figure;
(4) mass spectrometric data obtaining is processed with Mascot Distiller software, then searched in SwissProt database with MASCOT, setting parameter is as follows:
The maximum leakage of enzyme cut site: 2;
Fixing modification: Carbamidomethylation, halfcystine;
Variable modification :+HexNAc, 203Da, serine and threonine; + 0.9840Da, asparagine becomes aspartic acid; Oxidation, methionine; Acetylation, N-end; Cyclisation, N-end;
Parent ion quality error is 15ppm, and secondary fragment quality error is 0.8Da; By searching storehouse result, obtain protein glycosylation site information.
Preferred according to the present invention, in described step (1), restriction endonuclease is: trypsase or protein incision enzyme Glu-C (staphylococcus aureus V8), the mass ratio of restriction endonuclease and protein sample is 1:(20～30).
Preferred according to the present invention, in described step (1), endoglycosidase is: Peptide N-glycosidase F (PNGase F).
Preferred according to the present invention, in described step (1), exoglycosidase is: the mixing of β (1-3,4) galactosidase, β-NAG glycosides enzyme and sialidase.Further preferred, sample or the single protein sample of every 100 μ g after endonuclease digestion adds the amount of every kind of endoglycosidase and exoglycosidase to be 1 μ l, the concentration of described β (1-3,4) galactosidase is >=concentration that the concentration of 2U/ml, β-NAG glycosides enzyme is >=40U/ml, sialidase is >=5U/ml.
Preferred according to the present invention, in described step (1), the enzyme tangent condition of endoglycosidase and exoglycosidase is: under 37 ℃ of conditions, enzyme is cut 24h.
Preferred according to the present invention, in described step (2), durian agglutinin chromatographic column is prepared as follows: the Ago-Gel of the coupling durian agglutinin of 1.7mL is loaded in the perfluoroalkoxy resin pipe of 1 * 1900mm, coupling durian agglutinin concentration is 3.5～4.5mg/ml, makes durian agglutinin chromatographic column.
Preferred according to the present invention, the separation condition of durian agglutinin chromatographic column is as follows in described step (2): flow velocity 100 μ L/min, and with 8 column volumes of lavation buffer solution wash-out, then with 5 column volumes of elution buffer wash-out that contain 0.8M galactose; After eluent is collected, with the desalination of HyperSep C18 post, obtain;
Described lavation buffer solution is 100mM Tris – HCl, and pH 7.4.
Preferred according to the present invention, C in described step (3)
18the mobile phase A of reverse-phase chromatographic column is the solution that contains formic acid and acetonitrile, and formic acid mass concentration is 0.1%, and acetonitrile mass concentration is 2%; Mobile phase B is the solution that contains formic acid and acetonitrile, and formic acid mass concentration is 0.1%, and acetonitrile mass concentration is 98%.
Preferred according to the present invention, C in described step (3)
18reverse-phase chromatographic column testing conditions as follows:
The enzyme that step (1) is made is cut the group protein sample that rear single protein sample or step (2) make and is dissolved in mobile phase A, and being mixed with concentration is the solution to be measured of 0.5～1.5 μ g/ μ L;
Flow velocity 50 μ L/min, gradient is: 0～5min, 98% mobile phase A, 2% Mobile phase B; 5～25min, 85～98% mobile phase A, 2～15% Mobile phase B, 25～55min, 60～85% mobile phase A, 15～40% Mobile phase B, 55～60min, 2～60% mobile phase A, 40～98% Mobile phase B, 60～70min, 2% mobile phase A, 98% Mobile phase B.
Preferred according to the present invention, in described step (3), high resolution mass spectrum adopts LTQ-Orbitrap Velos Pro type high resolution mass spectrum, and setup parameter is: isotopic resolution: 60000; Quality of scanning scope: 400～1800; Data acquisition adopts data dependence pattern; Collision energy is set as 35%.
Beneficial effect
The present invention has set up the detection method of the O-glycosylation site of glycoprotein in single albumen and group protein sample, not only simplified operation steps, make the glycosylation site analysis of single albumen more simple and quick, and the method is learned in the system of the complexity such as protein sample in group, also can more fast, more accurately, more fully obtain site information, for the detection of O-glycosylation site in the standard model relevant to medical diagnosis on disease and actual sample, there is great practical value.
Accompanying drawing explanation
Fig. 1 Fig. 1 is the experiment flow of the method for the invention;
Fig. 2 myosin O-glycosylation site second order spectrum example;
The new O-glycosylation site of Fig. 3 hCG second order spectrum example;
Fig. 4 agglutinin pillar enrichment plasma proteins chromatogram and each flow point TIC figure;
The second order spectrum of Fig. 5 plasma proteins Proteoglycan 4 peptide section SPDESTPELSAEPTPK O-glycosylation sites;
Embodiment
Below in conjunction with embodiment, the present invention will be further described, but institute of the present invention protection domain is not limited to this.
Liquid chromatograph is that Shimadzu receives and rises liquid chromatograph; Mass spectrum is Thermo LTQ-Orbitrap Velos Pro type high resolution mass spectrum, and workstation is Xcalibur.
Embodiment 1
The method of myosin O-glycosylation site, step is as follows:
1.1 add 100mM to contain the ammonium bicarbonate buffers (pH8.2) of 6M guanidine hydrochloride in 50 μ g myosin samples, 1M dithiothreitol (DTT) (final concentration is 100mM), 37 ℃ of water-baths are hatched after 1h, add 1M iodoacetamide (final concentration is 150mM) room temperature lucifuge reaction 30min.Reactant liquor, with after the ultra filtration membrane ultrafiltration of 10kDa, adds trypsase (Trypsin, final concentration 1-2%w/w), and 37 ℃ are spent the night, and 100 ℃ of heating 2min cessation reactions, make endonuclease digestion product.
1.2 add Peptide N-glycosidase F 1 μ l by every 100 μ g endonuclease digestion products, β (1-3,4) galactosidase 1 μ l, β-NAG glycosides enzyme 1 μ l and sialidase A 1 μ l, 37 ℃ of enzymes are cut 24h, and then drying under reduced pressure, makes part desugar chain peptide section sample;
The acetonitrile solution of 1.3 2wt% that contain 0.1wt% formic acid is as mobile phase A;
The acetonitrile solution of 1.4 98wt% that contain 0.1wt% formic acid is as Mobile phase B;
1.5 are dissolved in mobile phase A by part desugar chain peptide section sample, and being mixed with concentration is the solution to be measured of 1 μ g/ μ L, use nano-LC system (Shimadzu) to carry out separation, sample C
18trapping column (Chemicals Evaluation and Research Institute, Japan) is carried out online desalination, then uses Reprosil-Pur C
18the C that beads (3 μ m) filler is loaded
18reverse-phase chromatographic column (15cm * 75 μ m i.d.) carries out the separation of peptide section; Flow velocity 50 μ L/min, gradient is: 0～5min, 98% mobile phase A, 2% Mobile phase B; 5～25min, 85～98% mobile phase A, 2～15% Mobile phase B, 25～55min, 60～85% mobile phase A, 15～40% Mobile phase B, 55～60min, 2～60% mobile phase A, 40～98% Mobile phase B, 60～70min, 2% mobile phase A, 98% Mobile phase B;
1.6 use Thermo LTQ OrbitrapVelos Pro mass spectrometer to detect under positive ion mode, obtain high resolution mass spectrum figure; Setup parameter is: isotopic resolution: 60000; Quality of scanning scope: 400～1800; Data acquisition adopts data dependence pattern; Collision energy is set as 35%.
1.7 process the mass spectrometric data obtaining with Mascot Distiller software, then search in SwissProt database with MASCOT, and setting parameter is as follows: species: mammal; Enzyme: trypsase; The maximum leakage of enzyme cut site: 2; Fixing modification: Carbamidomethylation (halfcystine); Variable modification :+HexNAc (203Da) (serine and threonine) ,+0.9840Da (asparagine becomes aspartic acid), oxidation (methionine), acetylation (N-end), cyclisation (N-end); Parent ion quality error is 15ppm, and secondary fragment quality error is 0.8Da.By searching storehouse result, obtain myosin glycosylation site information, as shown in table 1:
Table 1
The second order spectrum example that myosin is searched the glycosylation site that must fall in storehouse as shown in Figure 2.The O-glycosylation site of all bibliographical informations of myosin by this method, successfully detected.
Embodiment 2
Identical with the analytical approach of embodiment 1, difference is that analyzed sample is the short gland sex hormone (hCG) of human chorionic, as shown in table 2 by searching the hCG protein glycosylation site information that storehouse result obtains:
Table 2
Note: overstriking represents new discovery glycosylation site
The O glycosylation site of all bibliographical informations of hCG albumen by this method, not only successfully detected, and found 3 new glycosylation sites, its second order spectrum as shown in Figure 3.
Embodiment 3
The method of plasma proteins O-glycosylation site, step is as follows:
1.1 remove albumin by 20 μ L blood plasma to specifications with ProteoExtract Albumin Removal Kit
1.2 add 100mM to contain the ammonium bicarbonate buffers (pH8.2) of 6M guanidine hydrochloride by removing albuminous plasma sample, 1M dithiothreitol (DTT) (final concentration is 100mM), after 37 ℃ of water-bath 1h, add 1M iodoacetamide (final concentration is 150mM) room temperature lucifuge to process 30min.Reactant liquor, with after the ultra filtration membrane ultrafiltration of 10kDa, adds trypsase (Trypsin, final concentration 1-2%w/w), and 37 ℃ are spent the night, and 100 degree heating 2min cessation reactions, make endonuclease digestion product.
1.2 add Peptide N-glycosidase F 1 μ l by every 100 μ g endonuclease digestion products, β (1-3,4) galactosidase 1 μ l, β-NAG glycosides enzyme 1 μ l and sialidase A 1 μ l, 37 ℃ of enzymes are cut 24h, then drying under reduced pressure, makes with the peptide section of GalNAc sugar label and the sample of non-glycopeptide;
1.3 are loaded on the agarose of the coupling durian agglutinin of 1.7mL in the perfluoroalkoxy resin pipe of 1 * 1900mm, prepare durian agglutinin chromatographic column;
1.4 are splined on durian agglutinin chromatographic column with the GalNAc sugar peptide section of label and the sample of non-glycopeptide and carry out separated what make, flow velocity 100 μ L/min, with lavation buffer solution (100mM Tris – HCl, pH 7.4) 8 column volumes of wash-out, then with containing 5 column volumes of elution buffer wash-out of 0.8M galactose; After eluent is collected, use HyperSep C
18post desalination, then drying under reduced pressure, makes the sample being rich in GalNAc sugar labelled peptide section;
1.5 2% the acetonitriles that contain 0.1% formic acid are as mobile phase A;
1.6 98% the acetonitriles that contain 0.1% formic acid are as Mobile phase B;
1.7 are dissolved in mobile phase A by the sample being rich in GalNAc sugar labelled peptide section after enrichment, and making concentration is the solution to be measured of 1 μ g/ μ L, uses nano-LC system (Shimadzu) to carry out separation, sample C
18trapping column (Chemicals Evaluation and Research Institute, Japan) is carried out online desalination, then uses Reprosil-Pur C
18the C18 reverse-phase chromatographic column (15cm * 75 μ m i.d.) that beads (3 μ m) filler is loaded carries out the separation of peptide section; Flow velocity 50 μ L/min, gradient is: 0～5min, 98% mobile phase A, 2% Mobile phase B; 5～25min, 85～98% mobile phase A, 2～15% Mobile phase B, 25～55min, 60～85% mobile phase A, 15～40% Mobile phase B, 55～60min, 2～60% mobile phase A, 40～98% Mobile phase B, 60～70min, 2% mobile phase A, 98% Mobile phase B;
1.6 use Thermo LTQ OrbitrapVelos Pro mass spectrometer to detect under positive ion mode, obtain high resolution mass spectrum figure; Setup parameter is: isotopic resolution: 60000; Quality of scanning scope: 400～1800; Data acquisition adopts data dependence pattern; Collision energy is set as 35%.
1.7 process the mass spectrometric data obtaining with Mascot Distiller software, then search in SwissProt database with MASCOT, and setting parameter is as follows: species: people; Enzyme: Trypsin; The maximum leakage of enzyme cut site: 2; Fixing modification: Carbamidomethylation (halfcystine); Variable modification :+HexNAc (203Da) (serine and threonine) ,+0.9840Da (asparagine becomes aspartic acid), oxidation (methionine), acetylation (N-end), cyclisation (N-end); Parent ion quality error is 15ppm, and secondary fragment quality error is 0.8Da.By searching storehouse result, obtain plasma proteins glycosylation site information, as shown in table 3:
Table 3
Note: italics represents newfound glycoprotein or glycopeptide; Overstriking represents newfound glycosylation site
As shown in Figure 4, the second order spectrum example of plasma proteins O-glycosylation site as shown in Figure 5 for agglutinin pillar enrichment chromatogram and each flow point TIC figure.
Claims (10)
1. a method for analyzing proteins O-glycosylation site, is characterized in that, step is as follows:
(1) by protein group sample or single protein sample after endonuclease digestion, again through endoglycosidase and exoglycosidase combination excision N sugar chain and part O-sugar chain, through drying under reduced pressure, make with the GalNAc sugar peptide section of label and the group protein sample of non-glycopeptide or single protein sample;
(2) what step (1) is made is splined on durian agglutinin chromatographic column with the GalNAc sugar peptide section of label and the group protein sample of non-glycopeptide and carries out separated, after collecting enzyme and cutting with the peptide section solution of GalNAc sugar label, after drying under reduced pressure, make group and learn protein sample;
(3) the group protein sample that single protein sample step (1) being made or step (2) make is used C
18reverse-phase chromatographic column is separated, then under positive ion mode, with high-resolution mass spectrometer, detects, and obtains high resolution mass spectrum figure;
(4) mass spectrometric data obtaining is processed with Mascot Distiller software, then searched in SwissProt database with MASCOT, setting parameter is as follows:
The maximum leakage of enzyme cut site: 2;
Fixing modification: Carbamidomethylation, halfcystine;
Variable modification :+HexNAc, 203Da, serine and threonine; + 0.9840Da, asparagine becomes aspartic acid; Oxidation, methionine; Acetylation, N-end; Cyclisation, N-end;
Parent ion quality error is 15ppm, and secondary fragment quality error is 0.8Da; By searching storehouse result, obtain protein glycosylation site information.
2. the method for claim 1, is characterized in that, in described step (1), restriction endonuclease is: trypsase or protein incision enzyme Glu-C, the mass ratio of restriction endonuclease and protein sample is 1:(20～30).
3. the method for claim 1, is characterized in that, in described step (1), endoglycosidase is: Peptide N-glycosidase F.
4. the method for claim 1, is characterized in that, in described step (1), exoglycosidase is: the mixing of β (1-3,4) galactosidase, β-NAG glycosides enzyme and sialidase A; Further preferred, sample or the single protein sample of every 100 μ g after endonuclease digestion adds the amount of every kind of endoglycosidase and exoglycosidase to be 1 μ l, the concentration of described β (1-3,4) galactosidase is >=concentration that the concentration of 2U/ml, β-NAG glycosides enzyme is >=40U/ml, sialidase is >=5U/ml.
5. the method for claim 1, is characterized in that, in described step (1), the enzyme tangent condition of endoglycosidase and exoglycosidase is: under 37 ℃ of conditions, enzyme is cut 24h.
6. the method for claim 1, it is characterized in that, in described step (2), durian agglutinin chromatographic column is prepared as follows: the Ago-Gel of the coupling durian agglutinin of 1.7mL is loaded in the perfluoroalkoxy resin pipe of 1 * 1900mm, coupling durian agglutinin concentration is 3.5～4.5mg/ml, makes durian agglutinin chromatographic column.
7. the method for claim 1, it is characterized in that, the separation condition of durian agglutinin chromatographic column is as follows in described step (2): flow velocity 100 μ L/min, and with 8 column volumes of lavation buffer solution wash-out, then with 5 column volumes of elution buffer wash-out that contain 0.8M galactose; After eluent is collected, with the desalination of HyperSep C18 post, obtain;
Described lavation buffer solution is 100mM Tris – HCl, and pH 7.4.
8. the method for claim 1, is characterized in that, C in described step (3)
18the mobile phase A of reverse-phase chromatographic column is the solution that contains formic acid and acetonitrile, and formic acid mass concentration is 0.1%, and acetonitrile mass concentration is 2%; Mobile phase B is the solution that contains formic acid and acetonitrile, and formic acid mass concentration is 0.1%, and acetonitrile mass concentration is 98%.
9. the method for claim 1, is characterized in that, C in described step (3)
18reverse-phase chromatographic column testing conditions as follows:
The enzyme that step (1) is made is cut the group protein sample that rear single protein sample or step (2) make and is dissolved in mobile phase A, and being mixed with concentration is the solution to be measured of 0.5～1.5 μ g/ μ L;
Flow velocity 50 μ L/min, gradient is: 0～5min, 98% mobile phase A, 2% Mobile phase B; 5～25min, 85～98% mobile phase A, 2～15% Mobile phase B, 25～55min, 60～85% mobile phase A, 15～40% Mobile phase B, 55～60min, 2～60% mobile phase A, 40～98% Mobile phase B, 60～70min, 2% mobile phase A, 98% Mobile phase B.
10. the method for claim 1, is characterized in that, in described step (3), high resolution mass spectrum adopts LTQ-Orbitrap Velos Pro type high resolution mass spectrum, and setup parameter is: isotopic resolution: 60000; Quality of scanning scope: 400～1800; Data acquisition adopts data dependence pattern; Collision energy is set as 35%.
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